Delivery of therapeutic biologicals from implantable tissue matrices

ABSTRACT

Normal cells, such as fibroblasts or other tissue or organ cell types, are genetically engineered to express biologically active, therapeutic agents, such as proteins that are normally produced in small amounts, for example, MIS, or other members of the TGF-beta family Herceptin™, interferons, andanti-angiogenic factors. These cells are seeded into a matrix for implantation into the patient to be treated. Cells may also be engineered to include a lethal gene, so that implanted cells can be destroyed once treatment is completed. Cells can be implanted in a variety of different matrices. In a preferred embodiment, these matrices are implantable and biodegradable over a period of time equal to or less than the expected period of treatment, when cells engraft to form a functional tissue producing the desired biologically active agent. Implantation may be ectopic or in some cases orthotopic. Representative cell types include tissue specific cells, progenitor cells, and stem cells. Matrices can be formed of synthetic or natural materials, by chemical coupling at the time of implantation, using standard techniques for formation of fibrous matrices from polymeric fibers, and using micromachining or microfabrication techniques. These devices and strategies are used as delivery systems via standard or minimally invasive implantation techniques for any number of parenterally deliverable recombinant proteins, particularly those that are difficult to produce in large amounts and/or active forms using conventional methods of purification, for the treatment of a variety of conditions that produce abnormal growth, including treatment of malignant and benign neoplasias, vascular malformations (hemangiomas), inflammatory conditions, keloid formation, abdominal or plural adhesions, endometriosis, congenital or endocrine abnormalities, and other conditions that can produce abnormal growth such as infection. Efficacy of treatment with the therapeutic biologicals is detected by determining specific criteria, for example, cessation of cell proliferation, regression of abnormal tissue, or cell death, or expression of genes or proteins reflecting the above.

This application claims priority to U.S. Ser. No. 60/178,842 filed Jan.27, 2000.

The United States government has certain rights in this invention byvirtue of Grant No. CA17393 from the National Institutes of Health toPatricia K. Donahoe and David T. MacLaughlin; National Institute ofHealth grant No. CA71345; and Department of Defense 1200-202487 toJoseph P. Vacanti.

BACKGROUND OF THE INVENTION

The present invention is generally in the area of methods and systemsfor treatment of disorders such as cancer with biologically activeagents produced naturally by cells in extremely small quantities, usinggenetically engineered host cells or natural cells that secrete asubstance naturally implanted in biodegradable polymeric matrices.

One of the difficulties in treatment of conditions such as cancer usingprotein or other biological modifiers is the need for large quantitiesof the therapeutic agent to be delivered over an extended period oftime. For most of the compounds discovered during research on complexpathways or unique tissues, it has not been possible, or has not beencommercially feasible, to produce the compounds in sufficient quantityto treat the disorders. Numerous examples of these compounds, especiallyproteins, have been reported. One prominant example is angiostatin, anaturally occurring anti-angiogenic peptide identified by researchers atChildren's Medical Center in Boston, Mass. Although extremely promisingin mice (O'Reilly et al., Science 285(5435):1926-8 1999), the inabilityof the developers to produce large quantities of the peptide has provento be a major stumbling block to conducting clinical trials fortreatment of cancer.

Mullerian Inhibiting Substance (MIS) is another biological with greatpotential for treatment of cancer. MIS is produced by the fetal testisand causes the regression in males of the Müillerian duct, theforerunner of the female reproductive ducts. MIS has been shown to havegreat potential as a treatment for ovarian carcinomas (Chin et al,Cancer Research. 51:2101-2106, 1991; Masiakos, et al, Clinical CancerResearch, 5(11):3488-99 1999) which are derived from embryonic Müllerianstructures. Recombinant human MIS (rhMIS) produced in Chinese hamsterovary cells (CHO) in multiple roller bottles has antiproliferativeactivity against several human carcinoma cell lines (Chin, et al, 1991).Recently, it was also reported that rhMIS specifically binds to afunctional heteromeric serine threonine (Teixeira, et al., Androl.17(4):336-41 1996; Teixeira et al., Endocrinology Jan;137(1):160-5 1996)receptor on the surface of human ovarian cancer ascites cells andinhibits the growth in vitro of these cells and of cells obtaineddirectly from women with Stage III and IV disease (Masiakos et al.,1999). See also, U.S. Pat. Nos. 4,404,199, 4,487,833, 4,510,131,4,753,794, 4,792,601, 5,011,687, 5,198,420 and 5,661,126 to Donahoe, etal., the teachings of which are incorporated by reference herein.

The Pediatric Surgical Research Laboratories has tested the hypothesisthat MIS will be a useful therapeutic agent for certain epithelialovarian cancers in a number of in vitro studies described below, butonly limited trials have been conducted in vivo. A major obstacle hasbeen purifying sufficient recombinant protein of suitable potency andhomogeneity for patient use.

Late stage epithelial ovarian cancer is a common and highly lethalgynecologic malignancy. Despite advances in treatment over the past twodecades substantial improvement in overall survival has been slow andincremental and a high mortality remains. The coelomic epithelium, whichinvaginates to form the Müllerian duct, is also the origin of thesehighly lethal human ovarian cancers. The hypothesis that MIS is atherapeutic for these Müllerian derived tumors is predicated on previousobservations in which partially purified bovine MIS (Donahoe et al., JSurg Res. 23: 141-8, 1977) suppressed growth of a single human ovariancancer cell line in monolayer culture (Donahoe et al., Science.205:913-5, 1979), in stem cell assays (Fuller et al., J Clin EndocrMetab. 54:1051-5, 1982), and in vivo in nude mice (Donahoe et al., AnnSurgery. 194:472-80,1981). Additionally, bovine MIS inhibited the growthof a large number of primary ovarian, Fallopian, and uterine carcinomasobtained directly from patients and tested in colony inhibition assaysin soft agar (Fuller et al., Gynecol. Oncol. 22:135-148, 1985). Afterpurifying bovine MIS (Budzik et. al., Cell 34: 307-314, 1983), thebovine and human MIS cDNAs and genomic human MIS (Cate et al., Cell. 45:685-98 1986) were cloned. The human gene was used to produce highlypurified recombinant human MIS (rhMIS) (Cate et. al., Cold Spring HarborSymp Quant Biol 51 Pt 1:641-7 1986; MacLaughlin et al., Methods Enzymol.198: 358-69, 1991) to which monoclonal and polyclonal antibodies wereraised for use in a sensitive ELISA (Hudson et al., J Clin EndocrinolMetab. 70: 16-22, 1990; Lee et al J Clin Endocrinol Metab. 81:571-69,1996). The rhMIS, which is now produced in a series of roller bottlesand purified from the media (Ragin et al, Protein Expression andPurification, 1992; 3(3):236-45), was shown to inhibit three humancarcinoma cell lines of Müllerian origin (Chin et al., 1991), as well asa human ocular melanoma cell line (Parry et al., Cancer Res. 52:1182-6,1992), in vitro and in vivo, in a dose dependent manner (Chin et al.,1991; Boveri et al., Int J Oncology. 2; 135-44, 1993). In order to scaleup production beyond the roller bottle capacity which suites academicneeds, a clonal line of MIS-producing transfected CHO cells (CHO, B9)was transferred to CHO B9 seeded bioreactors, for scale up to completethe phase I trials. Purification protocols for the bioreactor producedprotein have been designed (Ragin et al, 1992), but modifications toimprove purification protocols to enhance recovery and cioactivity havenot resulted in production of sufficient quantities.

It is important to note that like the other members of the TGFβ family,the bioactive purified protein is not a single polypeptide chain but aproteolytically cleaved molecule. MIS is primarily processed at residue427, producing 110 kDa amino-terminal and 25 kDa carboxyterminaldisulfide bond reduction sensitive homodimers (Pepinsky et al, J. Biol.Chem. 1988; 263:18961-4.; MacLaughlin et al, EndocrinologyJul;131(1):291-6 1992). Although the carboxy terminus is the activedomain of rhMIS in vitro, it has not been shown to be active in vivo.The non-covalent association of the amino and carboxy termini ispresumed to prevent the rapid clearance characteristic of the C terminusin vivo. Therefore, MIS to be administered to patients is being producedas a cleaved but non-dissociated complex. Unfortunately, theimmunoaffinity purification protocols result in aggregation, given thehydrophobic character of MIS, with a product of reduced potency and lowyield, making a process consistent with general manufacturing practicesproblematic. Moreover, alternative systems which are more efficient forlarge scale in vitro production, such as bacterial, yeast, or insectcell expression systems, have not been successful for the production ofbiologically active preparations of MIS.

It is therefore an object of the present invention to provide methodsand reagents for production of clinically effective amounts oftherapeutic biologicals, especially proteins such as MIS, in vivo.

It is a further object of the present invention to provide methods andreagents for production of biologics in vivo, where the production canbe discontinued if appropriate.

It is a still further object of the present invention to provide methodsand reagents for treatment of a variety of disorders characterized bythe proliferation of abnormal tissue, including malignant and benignneoplasias, vascular malformations, inflammatory conditions includingrestenosis, infection, keloid formation and adhesions, congenital orendocrine abnormalities and other conditions that produce abnormalgrowth.

SUMMARY OF THE DISCLOSURE

Normal cells, such as fibroblasts or other tissue or organ cell types,are genetically engineered to express biologically active, therapeuticagents, such as proteins that are normally produced in small amounts,for example, MIS, Herceptin™, interferons, and Endostatin™, or naturallyproduced compounds. These cells are seeded into a matrix forimplantation into the patient to be treated. Cells may also beengineered to include a lethal gene, so that implanted cells can bedestroyed once treatment is completed. Cells can be implanted in avariety of different matrices. In a preferred embodiment, these matricesare implantable and biodegradable over a period of time equal to or lessthan the expected period of treatment, during which the engrafted cellsform a functional tissue producing the desired biologically active agentfor longer periods of time. Representative cell types include tissuespecific cells, progenitor cells, and stem cells. Matrices can be formedof synthetic or natural materials, by chemical coupling at the time ofimplantation, using standard techniques for formation of fibrousmatrices from polymeric fibers, and using micromachining ormicrofabrication techniques.

These devices and strategies are used as delivery systems, which may beimplanted by standard or minimally invasive implantation techniques, forany number of parenterally deliverable recombinant proteins,particularly those that are difficult to produce in large amounts and/oractive forms using conventional methods of purification, for thetreatment of a variety of conditions that produce abnormal growth,including treatment of malignant and benign neoplasias, vascularmalformations (hemangiomas), inflammatory conditions, keloid formationand adhesion, endometriosis, congenital or endocrine abnormalities, andother conditions that can produce abnormal growth such as infection.Efficacy of treatment with the therapeutic biologicals is detected bydetermining specific criteria, for example, cessation of cellproliferation, regression of abnormal tissue, or cell death.

The examples demonstrate the use of this method with a tissue specificbiological modifier, MIS. Genetically engineered CHO cells were grown onimplantable polymeric meshes and the levels of secreted rMIS measured.The polymeric meshes with CHO cells seeded therein were then implantedin vivo and serum levels of rMIS measured. Data show very high levels ofrMIS over prolonged time periods. These animals were then implanted withhuman ovarian cell lines and tumor regression measured in the presenceof the MIS-producing cells. The implanted cells significantly inhibitedthe tumor cell proliferation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of MIS (ng/day) production over days of incubation ofCHO B9 cells impregnated on mesh in vitro.

FIGS. 2A-C are graphs of MIS production on polymeric implants in mice.

FIG. 2A is a graph showing that the accumulation of MIS in serum isinfluenced by the size of the polymer implant. Polymer squares wereseeded with MIS-producing CHO-B9 cells 3 days prior to implantation.After 21 days, marked differences in the mean serum levels of MIS (n=4per polymer size) can be seen in the animals implanted with polymers ofvarying sizes. 0.5 cm² grafts were used for further study.

FIG. 2B is a graph showing increasing levels of MIS were detected byELISA on day 7, 14, 21, and 28 in the serum of SCID mice after theCHO-B9 seeded polymer was implanted in the right ovarian pedicle. Thiscomposite figure shows the mean +/− the standard error of the mean for atotal of 40 animals.

FIG. 2C is a graph showing that the accumulation of MIS in the serum ofpolymer-bearing mice is reversible. Two weeks after implantation of apolymer-cell graft into the right ovarian pedicle of a SCID mouse theserum level of MIS was approximately 1200 ng/ml. Upon removal of thepolymer, the MIS falls to undetectable levels, consistent with lack ofmigration of MIS-producing cells from the site of implantation (n=1).

FIGS. 3A and 3B are graphs of MIS inhibition of the human ovarian tumorcell line IGROV-1 tumor growth in animals implanted with CHO B9 cellsseeded on polyglycolic acid mesh as a function of graft size ratio (mean± SEM), compared to a mesh seeded with non-transfected L9 cells as anegative control. The graft-size ratio represents the size of the tumorat two weeks divided by the starting size of the tumor and represents ameasure of tumor growth. The L9 animals are represented by the squaresand the B9 animals by the diamonds. The dashed line indicates the mean ±the standard error.

FIG. 3A shows that the mean graft-size ratios (GSR) +/− the standarderror of the mean of the IGROV-1 tumors in the animals producingbioactive MIS (n=8) was significantly smaller than the IGROV-1 tumorsexposed to bioinactive MIS (n=10) (p value=0.016). A GSR of 1 indicatesno net growth.

FIG. 3B shows that the mean GSR of the IGROV-1 tumors implanted in theanimals with MIS-producing CHO-B9 polymer (n=30) was significantlysmaller than the IGROV-1 tumors implanted in the animals with emptypolymer (n=30) (p value<0.001).

DETAILED DESCRIPTION OF THE INVENTION

A strength of biological modifiers is that they impart specificity totreatment paradigms to allow for prolonged parenteral therapy, andeliminate many of the side effects and inconveniences associated withconventional therapies. Problems which are often encountered with thesemolecules include purification and enrichment. Most are manufactured inthe laboratory using recombinant technology. A small number are selectedfor scale up by the pharmaceutical industry. As an important step fortheir purification, they undergo rigorous purification schemes usingseparation methods that may alter their chemical characteristics. As isoften the case, the end product is a small percentage of the startingmaterial, and is frequently less potent. To obviate the loss of quantityand potency, a polymer scaffolding or matrix has been used toproliferate cells producing the biological modifiers. When this scaffoldor matrix is implanted into an organism, it becomes vascularized orotherwise connected to the vasculature, the seeded cells grow to fillthe scaffold, the biological modifiers are secreted directly into thebloodstream or adjacent cells, and, in a preferred embodiment, thescaffold is resorbed, leaving a new secretory tissue. Elimination of thepurification steps enhances yield and avoids the problems withcontamination, cost and loss of biological activity.

I. Materials for Production of Secretory Tissues

The materials required for production in vivo of biologically activemolecules include cells which produce the biologically active moleculesand matrices for proliferation of the engineered cells which can beimplanted in vivo to form new secretory tissues. In one embodiment,cells are obtained which already produce the desired biologicalmodifiers. In another embodiment, cells are genetically engineered toproduce the biological modifiers. In this embodiment, it is alsonecessary to provide the appropriate genes, means for transfection ofthe cells, and means for expression of the genes.

A. Cells to be Engineered

As a proof of principle, CHO cells permanently transfected by calciumphosphate precipitation with the MIS gene on a CMV promotor and clonallyselected for the highest MIS producers were used in preparations andimplantations. The devices were seeded with cells for 4-7 days prior toimplantation and MIS levels measured in the serum by a sensitive MISELISA. The next step was to implant non-tumor cells, such asfibroblasts, both cell lines and then the patient's own fibroblasts toavoid rejection. These cells likewise can be engineered to express, andsecrete the desired biological molecule(s). Other representative celltypes include other patient specific differentiated cells, progenitor orembryonic or pluripotential stem cells.

Cells to be engineered can be obtained from established cell culturelines, by biopsy or from the patient or other individuals of compatibletissue types. The preferred cells are those obtained from the patient tobe treated. In those cases where the patient's own cells are not used,the patient will also be treated with appropriate immunosuppressantssuch as cyclosporine to avoid destruction of the implanted cells duringtherapy.

In the preferred embodiments, cells are obtained directly from thedonor, washed, and cultured using techniques known to those skilled inthe art of tissue culture. Cells are then transfected with the gene ofinterest and seeded at various cell counts onto a matrix such as apolymeric mesh to achieve optimal production of a biological such asMIS.

Cell attachment and viability can be assessed using scanning electronmicroscopy, histology, and quantitative assessment, for example, byELISA, fluorescent labelled or radioactive labelled antibodies. Thefunction of the implanted cells can be determined using a combination ofthe above-techniques and functional assays. Studies using protein assayscan be performed to quantitate cell mass on the polymer scaffolds. Thesestudies of cell mass can then be correlated with cell functional studiesto determine the appropriate cell mass.

B. Biologically Active Molecules

Any biologically active molecule which has been cloned or for which acellular source is available can be used. Representative molecules arethose having a known activity which selectively reduces the symptoms ofthe disorder to be treated, such as MIS, Herceptin, interferons,endostatin, and growth factors such as tumor necrosis factor. Forexample, for the treatment of malignant or benign hyperplasia, thebiologically active molecules include anti-angiogenic compounds, MIS andother hormones which selectively or preferentially bind to the cells tobe killed or inactivated. Alternatively, the cells can be engineered tocorrect the defect in the cells which results in overproliferation.

The goal is not to form a permanent new tissue, but to provide animplanted “bioreactor” to produce therapeutic biologicals for a definedperiod effective to cause cessation of cell proliferation, regression ofabnormal tissue, or cell death. Various devices and strategies are usedas delivery systems which can be transplanted by standard or byminimally invasive implantation techniques for any number ofparenterally deliverable recombinant proteins, particularly those thatare difficult to produce in large amounts and/or active forms usingconventional methods of purification, for the treatment of a variety ofconditions that produce abnormal growth, including treatment ofmalignant and benign neoplasias, vascular malformations (hemangiomas),inflammatory conditions, keloid formation, endometriosis, congenital orendocrine abnormalities, and other conditions that can produce abnormalgrowth such as infection.

C. Vectors for Engineering Cells

Examples of recombinant DNA techniques include cloning, mutagenesis, andtransformation. Recombinant DNA techniques are disclosed in Maniatis etal., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.(1982). Vectors, including adeno-associated viruses, adenoviruses,retroviruses, and tissue specific vectors, are commercially available.Vectors can include secretory sequences, so that the biological modifierwill diffuse out of the cell in which it is expressed and into thevascular supply or interstitial spaces in order to expose the cells ofinterest to concentrations of the protein that are effective to treatthe patient. The vector or expression vehicle, and in particular thesites chosen therein for insertion of the selected DNA fragment and theexpression control sequence, are determined by a variety of factors,e.g., number of sites susceptible to cleavage by a particularrestriction enzyme, size of the protein to be expressed, expressioncharacteristics such as start and stop codons relative to the vectorsequences, and other factors recognized by those of skill in the art.The choice of a vector, expression control sequence, and insertion sitefor DNA sequence encoding the biological modifier is determined by abalance of these factors.

It should be understood that the DNA sequences coding for the biologicalmodifier that are inserted at the selected site of a cloning orexpression vehicle may include nucleotides which are not part of theactual gene coding for the biological modifier or may include only afragment of the actual gene. It is only required that whatever DNAsequence is employed, a transformed host cell will produce thebiological modifier. For example, MIS DNA sequences may be fused in thesame reading frame in an expression vector with at least a portion of aDNA sequence coding for at least one eukaryotic or prokaryotic signalsequence, or combinations thereof. Such constructions enable theproduction of, for example, a methionyl or other peptidyl-MISpolypeptide. This N-terminal methionine or peptide may either then becleaved intra- or extra-cellularly by a variety of known processes orthe MIS polypeptide with the methionine or peptide attached may be used,uncleaved.

The complete nucleotide and amino acid sequence for human and bovineMIS, and cloning and expression vehicles, are provided in U.S. Pat. No.5,047,336 to Cate, et al. and Cate et al., Cell 45:685-698(1986), andare also available using publicly available gene data bases andcommercial suppliers.

D. Matrices

There are three basic types of matrices that can be used: devices formedby micromachining, micromolding or other microfabrication techniques,fibrous polymeric scaffolds, and hydrogels.

1. Microfabricated Device Design and Manufacture

Preferred materials for making devices to be seeded with cells arebiodegradable polymers, although in some embodiments non-degradablematerials may be preferred or may be used as structural support or ascomponents of a device formed of biodegradable polymer. The polymercomposition can be selected both to determine the rate of degradation aswell as to optimize proliferation. Many biodegradable, biocompatiblepolymeric materials can be used to form the device, or guide channelswithin the device, including both natural and synthetic polymers, andcombinations thereof. Examples of natural polymers include proteins suchas collagen, collagen-glycosaminoglycan copolymers, polysaccharides suchas the celluloses (including derivatized celluloses such asmethylcelluloses), extracellular basement membrane matrices such asBiomatrix, and polyhydroxyalkanoates such as polyhydroxybutyrate (PHB)and polyhydroxybutyrate-co-valerate (PHBV) which are produced bybacterial fermentation processes. Synthetic polymers include polyesterssuch as polyhydroxyacids like polylactic acid (PLA), polyglycolic acid(PGA) and compolymers thereof (PLGA), some polyamides andpoly(meth)acrylates, and polyanhdyrides. Examples of non-degradablepolymers include ethylenevinylacetate (EVA), polycarbonates, and somepolyamides.

The surface morphology of the devices can affect cell growth. Bioactivematerials may also be incorporated into the device or a sustainedrelease matrix within the device to promote cell viability orproliferation. These materials can be incorporated into the polymer at aloading designed to release by diffusion and/or degradation of thepolymer forming the device over a desired time period, ranging from daysto weeks. Alternatively, the bioactive substance may be incorporatedinto a matrix loaded into or adjacent to the device. These matrices maybe formed of the same materials as the device or may consist ofpolymeric materials incorporated within the tracts or channels, forexample, hydrogel matrices of the types described in the literature (forexample, Wells, et al., Exp. Neurol. (1997) 146(2):395-402; Chamberlain,et al., Biomaterials 1998 19(15):1393-1403; and Woerly, et al., (1999)J. Tissue Engineering 5(5):467-488) for use in promoting nerve growth.Examples of such materials include polyamide, methylcellulose,polyethyleneoxide block compolymers such as the Pluronics, especiallyF127 (BASF), collagen, and extracellular matrix (ECM) of the type soldas Biomatrix. Other useful materials include the polymer foams reportedby Hadlock, et al., Laryngoscope (1999) 109(9):1412-1416.

Microfabrication techniques include micromachining, solid free form(SFF) techniques, and micromolding techniques, as well as othertechniques based on well-established methods used to make integratedcircuits, electronic packages and other microelectronic devices, havingdimensions as small as a few nanometers and which can be mass producedat low per-unit costs.

Micromachining Techniques

Micromachining techniques are described in the literature, for example,by Rai-Choudhury, ed. Handbook of Microlithography, Micromaching &Microfabrication (SPIE Optical Engineering Press, Bellingham, Wash.1997), the teachings of which are incorporated herein. The techniquescan be used to form the device directly, or as discussed below, to formmolds which are then used to form the devices.

Other microfabrication processes that may be used include lithography;etching techniques, such as wet chemical, dry, and photoresist removal;thermal oxidation; film deposition, such as evaporation (filament,electron beam, flash, and shadowing and step coverage), sputtering,chemical vapor deposition (CVD), epitaxy (vapor phase, liquid phase, andmolecular beam), electroplating, screen printing, lamination, lasermachining, and laser ablation (including projection ablation). Seegenerally Jaeger, Introduction to Microelectronic Fabrication(Addison-Wesley Publishing Co., Reading Mass. 1988); Runyan, et al.,Semiconductor Integrated Circuit Processing Technology (Addison-WesleyPublishing Co., Reading Mass. 1990); Proceedings of the IEEE MicroElectro Mechanical Systems Conference 1987-1998; Rai-Choudhury, ed.,Handbook of Microlithograhy Micromachining & Microfabrication (SPIEOptical Engineering Press, Bellingham, Wash. 1997).

Deep plasma etching can be used to create structures with diameters onthe order of 0.1 μm or larger. In this process, an appropriate maskingmaterial is deposited onto a substrate and patterned into dots havingthe diameter of the desired tracts or channels. The wafer is thensubjected to a carefully controlled plasma. Those regions protected bythe metal mask remain and form the tracts.

Another method for forming devices including tracts or channels is touse microfabrication techniques such as photolithography, plasmaetching, or laser ablation to make a mold form, transferring that moldform to other materials using standard mold transfer techniques, such asembossing or injection molding, and reproducing the shape of theoriginal mold form using the newly-created mold to yield the finaldevice. Alternatively, the creation of the mold form could be skippedand the mold could be microfabricated directly, which could then be usedto create the final device.

Micromolding Techniques

Another method of fabricating tracts or channels utilizes micromoldplating techniques. A photo-defined mold first is first produced, forexample, by spin casting a thick layer, typically 150 μm, of an epoxyonto a substrate that has been coated with a thin sacrificial layer,typically about 10 to 50 nm. Arrays of cylindrical holes are thenphotolithographically defined through the epoxy layer, which typicallyis about 150 μm thick. (Despont, et al., “High-Aspect-Ratio, Ultrathick,Negative-Tone Near-UV Photoresist for MEMS,” Proc. of IEEE 10^(th)Annual International Workshop on MEMS, Nagoya, Japan, pp. 518-522 (Jan.26-30, 1997)). The diameter of these cylindrical holes defines the outerdiameter of the tracts. The upper surface of the substrate, thesacrificial layer, is then partially removed at the bottom of thecylindrical holes in the photoresist. The exact method chosen depends onthe choice of substrate. For example, the process has been successfullyperformed on silicon and glass substrates (in which the upper surface isetched using isotropic wet or dry etching techniques) and copper-cladprinted wiring board substrates. In the latter case, the copper laminateis selectively removed using wet etching. Then a seed layer, such asTi/Cu/Ti (e.g., 30 nm/200 nm/30 nm), is conformally DC sputter-depositedonto the upper surface of the epoxy mold and onto the sidewalls of thecylindrical holes. The seed layer should be electrically isolated fromthe substrate. Subsequently, one or more electroplatable metals oralloys, such as Ni, NiFe, Au, Cu, or Ti, are electroplated onto the seedlayer. The surrounding epoxy is then removed, leaving molds which eachhave an interior annular hole that extends through the base metalsupporting the tracts. The rate and duration of electroplating iscontrolled in order to define the wall thickness and inner diameter ofthe tracts.

The molds made as described above and injection molding techniques canbe applied to form the tracts or channels in the molds (Weber, et al.,“Micromolding—a powerful tool for the large scale production of precisemicrostructures”, Proc. SPIE—International Soc. Optical Engineer. 2879,156-167 (1996); Schift, et al., “Fabrication of replicated highprecision insert elements for micro-optical bench arrangements” Proc.SPIE—International Soc. Optical Engineer. 3513, 122-134 (1998). Thesemicromolding techniques can provide relatively less expensivereplication, i.e. lower cost of mass production.

Solid Free Form Manufacturing Techniques

As defined herein, SFF refers to any manufacturing technique that buildsa complex three dimensional object as a series of two dimensionallayers. The SFF methods can be adapted for use with a variety ofpolymeric, inorganic, and composite materials to create structures withdefined compositions, strengths, and densities, using computer aideddesign (CAD).

Examples of SFF methods include stereo-lithography (SLA), selectivelaser sintering (SLS), ballistic particle manufacturing (BPM), fusiondeposition modeling (FDM), and three dimensional printing (3DP). In apreferred embodiment, 3DP is used to precisely create channels and poreswithin a matrix to control subsequent cell growth and proliferation inthe matrix of one or more cell types having a defined function, such asnerve cells.

The macrostructure and porous parameters can be manipulated bycontrolling printing parameters, the type of polymer and particle size,as well as the solvent and/or binder. Porosity of the matrix walls, aswell as the matrix per se, can be manipulated using SFF methods,especially 3DP. Structural elements that maintain the integrity of thedevices during erosion can also be incorporated. For example, to providesupport, the walls of the device can be filled with resorbable inorganicmaterial, which can further provide a source of mineral for theregenerating tissue. Most importantly, these features can be designedand tailored using computer assisted design (CAD) for individualpatients to individualize the fit of the device.

Three Dimensional Printing (3DP).

3DP is described by Sachs, et al., “CAD-Casting: Direct Fabrication ofCeramic Shells and Cores by Three Dimensional Printing” ManufacturingReview 5(2), 117-126 (1992) and U.S. Pat. No. 5,204,055 to Sachs, etal., the teachings of which are incorporated herein. Suitable devicesinclude both those with a continuous jet stream print head and adrop-on-demand stream print head. A high speed printer of the continuoustype, for example, is the Dijit printer made and sold by Diconix, Inc.,of Dayton, Ohio, which has a line printing bar containing approximately1,500 jets which can deliver up to 60 million droplets per second in acontinuous fashion and can print at speeds up to 900 feet per minute.Both raster and vector apparatuses can be used. A raster apparatus iswhere the printhead goes back and forth across the bed with the jetturning on and off. This can have problems when the material is likelyto clog the jet upon settling. A vector apparatus is similar to an x-yprinter. Although potentially slower, the vector printer may yield amore uniform finish.

3DP is used to create a solid object by ink-jet printing a binder intoselected areas of sequentially deposited layers of powder. Each layer iscreated by spreading a thin layer of powder over the surface of a powderbed. The powder bed is supported by a piston which descends upon powderspreading and printing of each layer (or, conversely, the ink jets andspreader are raised after printing of each layer and the bed remainsstationary). Instructions for each layer are derived directly from acomputer-aided design (CAD) representation of the component. The area tobe printed is obtained by computing the area of intersection between thedesired plane and the CAD representation of the object. The individualsliced segments or layers are joined to form the three dimensionalstructure. The unbound powder supports temporarily unconnected portionsof the component as the structure is built but is removed aftercompletion of printing.

As shown in U.S. Pat. No. 5,204,055, the 3DP apparatus includes a powderdispersion head which is driven reciprocally in a shuttle motion alongthe length of the powder bed. A linear stepping motor assembly is usedto move the powder distribution head and the binder deposition head. Thepowdered material is dispensed in a confined region as the dispensinghead is moved in discrete steps along the mold length to form arelatively loose layer having a typical thickness of about 100 to 200microns, for example. An ink-jet print head having a plurality ofink-jet dispensers is also driven by the stepping motor assembly in thesame reciprocal manner so as to follow the motion of the powder head andto selectively produce jets of a liquid binder material at selectedregions which represent the walls of each cavity, thereby causing thepowdered material at such regions to become bonded. The binder jets aredispensed along a line of the printhead which is moved in substantiallythe same manner as the dispensing head. Typical binder droplet sizes arebetween about 15 to 50 microns in diameter. The powder/binder layerforming process is repeated so as to build up the device layer by layer.While the layers become hardened or at least partially hardened as eachof the layers is laid down, once the desired final part configuration isachieved and the layering process is complete, in some applications itmay be desirable that the form and its contents be heated or cured at asuitably selected temperature to further promote binding of the powderparticles. In either case, whether or not further curing is required,the loose, unbonded powder particles are removed using a suitabletechnique, such as ultrasonic cleaning, to leave a finished device.Finer feature size is also achieved by printing polymer solutions ratherthan pure solvents.

Stereo-lithography (SLA) and selective laser sintering (SLS).

SFF methods are particularly useful for their ability to controlcomposition and microstructure on a small scale for the construction ofthese medical devices. The SFF methods, in addition to 3DP, that can beutilized to some degree as described herein are stereo-lithography(SLA), selective laser sintering (SLS), ballistic particle manufacturing(BPM), and fusion deposition modeling (FDM).

Stereolithography is based on the use of a focused ultra-violet (UV)laser which is vector scanned over the top of a bath of aphotopolymerizable liquid polymer material. The UV laser causes the bathto polymerize where the laser beam strikes the surface of the bath,resulting in the creation of a first solid plastic layer at and justbelow the surface. The solid layer is then lowered into the bath and thelaser generated polymerization process is repeated for the generation ofthe next layer, and so on, until a plurality of superimposed layersforming the desired device is obtained. The most recently created layerin each case is always lowered to a position for the creation of thenext layer slightly below the surface of the liquid bath. A system forstereolithography is made and sold by 3D Systems, Inc., of Valencia,Calif., which is readily adaptable for use with biocompatible polymericmaterials.

SLS also uses a focused laser beam, but to sinter areas of a looselycompacted plastic powder, the powder being applied layer by layer. Inthis method, a thin layer of powder is spread evenly onto a flat surfacewith a roller mechanism. The powder is then raster-scanned with ahigh-power laser beam. The powder material that is struck by the laserbeam is fused, while the other areas of powder remain dissociated.Successive layers of powder are deposited and raster-scanned, one on topof another, until an entire part is complete. Each layer is sintereddeeply enough to bond it to the preceding layer. A suitable systemadaptable for use in making medical devices is available from DTMCorporation of Austin, Tex.

Ballistic particle manufacturing (BPM) and Fusion deposition modeling(FDM)

BPM uses an ink-jet printing apparatus wherein an ink-jet stream ofliquid polymer or polymer composite material is used to createthree-dimensional objects under computer control, similar to the way aninkjet printer produces two-dimensional graphic printing. The device isformed by printing successive cross-sections, one layer after another,to a target using a cold welding or rapid solidification technique,which causes bonding between the particles and the successive layers.This approach as applied to metal or metal composites has been proposedby Automated Dynamic Corporation of Troy, N.Y.

FDM employs an x-y plotter with a z motion to position an extrudablefilament formed of a polymeric material, rendered fluid by heat or thepresence of a solvent. A suitable system is available from Stratasys,Incorporated of Minneapolis, Minn.

Polymer Materials, Binders and Solvents for use in SSF Techniques

Depending on the processing method, the material forming the matrix maybe in solution, as in the case of SLA, or in particle form, as in thecase of SLS, BPM, FDM, and 3DP. In the preferred embodiment, thematerial is a polymer. In SLS, the polymer must be photopolymerizable.In the other methods, the material is preferably in particulate form andis solidified by application of heat, solvent, or binder (adhesive). Inthe case of SLS and FDM, it is preferable to select polymers havingrelatively low melting points, to avoid exposing incorporated bioactiveagent to elevated temperatures.

A number of materials are commonly used to form a matrix. Unlessotherwise specified, the term “polymer” will be used to include any ofthe materials used to form the matrix, including polymers and monomerswhich can be polymerized or adhered to form an integral unit, as well asinorganic and organic materials, as discussed below. In a preferredembodiment the particles are formed of a polymer which can be dissolvedin an organic solvent and solidified by removal of the solvent, such asa synthetic thermoplastic polymer, for example, ethylene vinyl acetate,poly(anhydrides), polyorthoesters, polymers of lactic acid and glycolicacid and other α hydroxy acids, polyhydroxyalkanoates, andpolyphosphazenes, a protein polymer, for example, albumin or collagen,or a polysaccharide. The polymer can be non-biodegradable orbiodegradable, typically via hydrolysis or enzymatic cleavage. Examplesof non-polymeric materials which can be used to form a part of thedevice or matrix for drug delivery include organic and inorganicmaterials such as hydoxyapatite, calcium carbonate, buffering agents,and lactose, as well as other common excipients used in drugs, which aresolidified by application of adhesive or binder rather than solvent. Inthe case of polymers for use in making devices for cell attachment andgrowth, polymers are selected based on the ability of the polymer toelicit the appropriate biological response from cells, for example,attachment, migration, proliferation and gene expression.

Photopolymerizable, biocompatible water-soluble polymers includepolyethylene glycol tetraacrylate (Mw 18,500) which can bephotopolymerized with an argon laser under biologically compatibleconditions using an initiator such as triethanolamine,N-vinylpyrrolidone, and eosin Y. Similar photopolymerizable macromershaving a poly(ethylene glycol) central block, extended with hydrolyzableoligomers such as oligo(d,1-lactic acid) or oligo(glycolic acid) andterminated with acrylate groups, may be used.

Examples of biocompatible polymers with low melting temperatures includepolyethyleneglycol 400 (PEG) which melts at 4-8° C., PEG 600 which meltsat 20-25° C., and PEG 1500 which melts at 44-48° C. Another low meltingmaterial is stearic acid, which melts at 70° C.

Other suitable polymers can be obtained by reference to The PolymerHandbook, 3rd edition (Wiley, N.Y., 1989), the teachings of which areincorporated herein.

A preferred material is a polyester in the polylactide/polyglycolidefamily. These polymers have received a great deal of attention in thedrug delivery and tissue regeneration areas for a number of reasons.They have been in use for over 20 years in surgical sutures, are Foodand Drug Administration (FDA)-approved and have a long and favorableclinical record. A wide range of physical properties and degradationtimes can be achieved by varying the monomer ratios in lactide/glycolidecopolymers: poly-L-lactic acid (PLLA) and poly-glycolic acid (PGA)exhibit a high degree of crystallinity and degrade relatively slowly,while copolymers of PLLA and PGA, PLGAs, are amorphous and rapidlydegraded.

Solvents and/or binder are used in the preferred method, 3DP, as well asSLA and BPM. The binder can be a solvent for the polymer and/orbioactive agent or an adhesive which binds the polymer particles.Solvents for most of the thermoplastic polymers are known, for example,methylene chloride or other organic solvents. Organic and aqueoussolvents for the protein and polysaccharide polymers are also known,although an aqueous solution, for example, containing a crosslinkingagent such as carbodiimide or glutaraldehyde, is preferred ifdenaturation of the protein is to be avoided. In some cases, however,binding is best achieved by denaturation of the protein.

The binder can be the same material as is used in conventional powderprocessing methods or may be designed to ultimately yield the samebinder through chemical or physical changes that take place in thepowder bed after printing, for example, as a result of heating,photopolymerization, or catalysis.

These methods and materials are further described in PCT/US96/09344“Vascularized Tissue Regeneration Matrices Formed by Solid Free-FormFabrication Methods” Massachusetts Institute of Technology andChildren's Medical Center Corporation.

2. Fibrous Scaffolds for Implantation

Fibrous scaffolding can be used to implant the cells, for example, asdescribed in U.S. Pat. No. 5,759,830 to Vacanti, et al. The design andconstruction of the scaffolding is of primary importance. The matrixshould be a pliable, non-toxic, porous template for vascular ingrowth.The pores should allow vascular ingrowth and the injection of cells intothe scaffold without damage to the cells or patient. The scaffolds aregenerally characterized by interstitial spacing or interconnected poresin the range of at least between approximately 100 and 300 microns indiameter. The matrix should be shaped to maximize surface area, to allowadequate diffusion of nutrients and growth factors to the cells and toallow the ingrowth of new blood vessels and connective tissue.

The same type of polymers can be used as in the Solid Free FormManufacturing techniques described above. In the preferred embodiment,the matrix is formed of a bioabsorbable, or biodegradable, syntheticpolymer such as a polyanhydride, polyorthoester, polyhydroxy acid suchas polylactic acid, polyglycolic acid, or a natural polymer likepolyalkanoates such as polyhydroxybutyrate and copolymers or blendsthereof. Proteins such as collagen can be used, but is not ascontrollable and is not preferred. These materials are all commerciallyavailable. Non-biodegradable polymers, including polymethacrylate andsilicon polymers, can be used, depending on the ultimate disposition ofthe growing cells.

In some embodiments, attachment of the cells to the polymer is enhancedby coating the polymers with compounds such as basement membranecomponents, agar, agarose, gelatin, gum arabic, collagens types I, II,III, IV, and V, fibronectin, laminin, glycosaminoglycans, mixturesthereof, and other materials, especially attachment peptides andpolymers having attachment peptides or other cell surface ligands boundthereto, known to those skilled in the art of cell culture. Vitrogen—100collagen (PCO 701) has been used in these experiments.

3. Hydrogel Matrices for Implantation

Polymeric materials which are capable of forming a hydrogel can beutilized. The polymer is mixed with cells for implantation into the bodyand is permitted to crosslink to form a hydrogel matrix containing thecells either before or after implantation in the body. In oneembodiment, the polymer forms a hydrogel within the body upon contactwith a crosslinking agent. A hydrogel is defined as a substance formedwhen an organic polymer (natural or synthetic) is crosslinked viacovalent, ionic, or hydrogen bonds to create a three-dimensionalopen-lattice structure which entraps water molecules to form a gel.Naturally occurring and synthetic hydrogel forming polymers, polymermixtures and copolymers may be utilized as hydrogel precursors. See forexample, U.S. Pat. No. 5,709,854 and WO 94/25080 by Reprogenesis.

In one embodiment, calcium alginate and certain other polymers that canform ionic hydrogels which are malleable. For example, a hydrogel can beproduced by cross-linking the anionic salt of alginic acid, acarbohydrate polymer isolated from seaweed, with calcium cations, whosestrength increases with either increasing concentrations of calcium ionsor alginate. The alginate solution is mixed with the cells to beimplanted to form an alginate suspension which is injected directly intoa patient prior to hardening of the suspension. The suspension thenhardens over a short period of time due to the presence in vivo ofphysiological concentrations of calcium ions. Modified alginatederivatives, for example, more rapidly degradable or which arederivatized with hydrophobic, water-labile chains, e.g., oligomers ofε-caprolactone, may be synthesized which have an improved ability toform hydrogels. Additionally, polysaccharides which gel by exposure tomonovalent cations, including bacterial polysaccharides, such as gellangum, and plant polysaccharides, such as carrageenans, may be crosslinkedto form a hydrogel using methods analogous to those available for thecrosslinking of alginates described above. Additional examples ofmaterials which can be used to form a hydrogel include polyphosphazinesand polyacrylates, which are crosslinked ionically, or block copolymerssuch as Pluronics™ or Tetronics™, polyethylene oxide-polypropyleneglycol block copolymers which are crosslinked by temperature or pH,respectively. Other materials include proteins such as fibrin (althoughthis is not preferred since thrombin may stimulate tumor growth via apathway that MIS may have to overcome, such as EGF-stimulatedproliferation), polymers such as polyvinylpyrrolidone, hyaluronic acidand collagen. Polymers such as polysaccharides that are very viscousliquids or are thixotropic, and form a gel over time by the slowevolution of structure, are also useful. For example, hyaluronic acid,which forms an injectable gel with a consistency like a hair gel, may beutilized. Modified hyaluronic acid derivatives are particularly useful.Polymer mixtures also may be utilized. For example, a mixture ofpolyethylene oxide and polyacrylic acid which gels by hydrogen bondingupon mixing may be utilized. In one embodiment, a mixture of a 5% w/wsolution of polyacrylic acid with a 5% w/w polyethylene oxide(polyethylene glycol, polyoxyethylene) 100,000 can be combined to form agel over the course of time, e.g., as quickly as within a few seconds.

Covalently crosslinkable hydrogel precursors also are useful. Forexample, a water soluble polyamine, such as chitosan, can becross-linked with a water soluble diisothiocyanate, such as polyethyleneglycol diisothiocyanate. The isothiocyanates will react with the aminesto form a chemically crosslinked gel. Aldehyde reactions with amines,e.g., with polyethylene glycol dialdehyde also may be utilized. Ahydroxylated water soluble polymer also may be utilized.

Alternatively, polymers may be utilized which include substituents whichare crosslinked by a radical reaction upon contact with a radicalinitiator. For example, polymers including ethylenically unsaturatedgroups which can be photochemically crosslinked may be utilized, asdisclosed in WO 93/17669. Additionally, water soluble polymers whichinclude cinnamoyl groups which may be photochemically crosslinked may beutilized, as disclosed in Matsuda et al.,ASAID Trans., 38:154-157(1992).

II. Methods for Engineering and Implantation of Cells

A. Disorders to be Treated

A variety of conditions that produce abnormal growth, includingtreatment of malignant and benign neoplasias, vascular malformations(hemangiomas), inflammatory conditions including those resulting frominfection, especially chronic or recalcitrant conditions such as thosein the sinuses or which are cystic, keloid formation, endometriosis,congenital or endocrine abnormalities such as testotoxicosis (Teixeiraet al, PNAS 1999) and other conditions that produce abnormal growth, canbe treated.

Examples of tumor cells that can be treated with MIS include primary andmetastatic growth of the following: ovarian adenocarcinomas, endometrialadenocarcinomas, cervical carcinomas, vulvar epidermoid carcinomas,ocular melanomas, prostate, breast, and germ cell tumors. As initiallydemonstrated with MIS transfected cells, this methodology can be usedfor delivery of a large number of proteins to control abnormal tissuegrowth, particularly other members of the TGFP family. Coupled withminimally invasive delivery systems, the biodegradable implantsproducing the therapeutic proteins from transfected autologous cells canbe introduced into a variety of sites to deliver therapeutics,particularly where a local effect is advantageous. This allows use of avariety of recombinant proteins without the need for complexpurification protocols.

B. Engineering of Cells

In the preferred embodiment, patient cells are transfected with the geneto be expressed, for example, rhMIS cDNA, to produce cells having stablyincorporated therein the DNA encoding the molecules to be expressed.Methods yielding transient expression, such as most adenoviral vectors,are not preferred. Stable transfectants are obtained by culturing andselection for expression of the encoded molecule(s). Those cells thatexhibit stable expression are seeded onto/into the appropriate matrixand then implanted using techniques such as those described in thefollowing examples.

C. Seeding of Matrices

The level of expression of the bioactive molecules is measured prior toimplantation to insure that an adequate number of cells is implanted. Ingeneral, the higher the number of cells implanted, the better. Cells arepreferably cultured initially in vitro, then implanted before the matrixdegrades but when the level of bioactive molecules is highest. Anexample of a suitable seeding density is between 1 and 10×10⁶ cells on amatrix with a surface area of 0.25 cm².

D. Implantation of Matrices

The devices are implanted into the patient at the site in need oftreatment using standard surgical techniques. In one embodiment, thedevice is constructed, seeded with cells, and cultured in vitro prior toimplantation. The cells are cultured in the device, tested for high MISproduction by ELISA, then implanted.

The technique described herein can be used for delivery of manydifferent cell types for different purposes. Other endocrine producingtransfectant cells can also be implanted. The matrix may be implanted inone or more different areas of the body to suit a particularapplication. Matrices with hepatocytes or other high oxygen organ cellsmay be implanted into the mesentery to insure a good blood supply. Sitesother than the mesentery for injection or implantation of cells includethe ovarian pedicle, subcutaneous tissue, retroperitoneum, properitonealspace, and intramuscular space. The use of ovarian pedicle for MISproducing implants cause ovary and fallopian tubes to adhere to implants(Kristjansen, et al., 1994).

The need for these additional procedures depends on the particularclinical situation.

III. EXAMPLES

The present invention will be further understood by reference to thefollowing non-limiting examples.

Example 1 Production of MIS in CHO Cells In Vitro

Materials and Methods

Polyglycolic acid fibers (approximately 12 microns diameter) obtainedfrom Albany International were cut into 0.5×0.5 cm squares. 1×10 10⁶ CHOB9 cells are seeded onto the mesh by the static seeding method. After 4hours, the mesh was transferred into a new dish containing 10 ml offresh media. Media MIS concentration was measured serially over sevendays by ELISA. Another CHO cell line, CHO L9, which is transfected witha mutated rhMIS gene that produces non-cleavable bioinactive protein,was placed on the mesh as a negative control for the implantexperiments. Samples were also placed in the organ culture assay todetermine MIS bioactivity.

Results

In preliminary in vitro experiments, CHO B9 cells seeded onto apolyglycolic acid matrix produced large (i.e. microgram) quantities ofbioactive MIS as determined by ELISA (Hudson et al., J Clin EndocrinolMetab. 70: 16-22, 1990) and by a standard organ culture bioassay(Donahoe et al., 1977) which detects regression of the Müllerian duct.The results are shown in FIG. 1. After eight days, more than 2000 ng MISwere produced.

Example 2 Regression of Tumors by MIS-producing Cells Seeded ontoPolymeric Matrices Implanted into Animals

As described in Example 1, in vitro CHO cells transfected with the humanMIS gene were seeded onto a polyglycolic acid matrix and produced largequantities (micrograms) of bioactive MIS as determined by ELISA and by astandard in vitro organ culture bioassay. A production rate of 400ng/day/device was determined, corresponding to a production rate percell of 3 pg/cell/day. By serial sampling it was determined that 7-8days incubation produced optimal bioactive MIS production by the meshimpregnated with the B9 clone (a cell line tranfected with the human MISgenomic sequence).

Studies were then undertaken to determine MIS production with this modelin vivo. The MIS producing matrices were implanted into the ovarianpedicle of B and T cell deficient 6-week-old female SCID mice. Serumlevels of MIS were measured to determine the rate of rise and durationof MIS production by the explants. Supraphysiologic levels of MIS weredetected in mouse sera within three days of implantation. It wasdetermined that the amount of MIS produced depends on the size of themesh implanted.

Several ovarian cancer cell lines were tested in vitro for inhibition byMIS. Human ovarian cancer cell lines (IGROV-1, OVCAR-8, OVCAR-5) wereplated on soft agarose and colony counts were determined as an assay endpoint. Significant inhibition (20-80%) of these ovarian cancer celllines by MIS was observed. Ovarian cancer cell lines that wereresponsive to MIS in vitro were then placed beneath the renal capsulesof SCID mice. These tumors grew two to four fold at two weeks afterimplantation with IGROV-1 showing the best growth. This represents ananimal model of tumor growth.

The inhibitory properties of the MIS produced on the mesh was thentested in vivo against the IGROV-1 tumor cell line. B9 and L9impregnated meshes were implanted in mice. The L9 cells producebio-inactive MIS and served as the negative control for the experiment.MIS produced by the B9 clones significantly inhibited the growth of thehuman ovarian cancer cell line in vivo, as follows.

Materials and Methods

Animals

Severe combined immunodeficient (SCID) female mice and athymic nude mice(6 weeks old, average weight 18-20 g) were obtained from and studied inthe Edwin L. Steele Laboratory, Massachusetts General Hospital, Boston,Mass. All animals were cared for and experiments performed in thisfacility under AAALAS approved guidelines using protocols approved bythe Institutional Review Board-Institutional Animal Care and UseCommittee of the Massachusetts General Hospital protocol #98-4254).

Cells

The IGROV-1 human ovarian cancer cell line was obtained from theAmerican Type Cell Culture and maintained in Dulbecco's Modification ofEagle's Medium (DMEM) supplemented with glutamine, penicillin,streptomycin, and 10% MIS-free female fetal calf serum. The cells weregrown at 37° C. in a humidified chamber perfused with 5% CO₂ in air. At80% confluency the cells were passed at a ratio of 1:4 and were carriedfor up to twelve passages.

The CHO-B9 cell line was formed by cloning the human MIS gene intodihydrofolate reductase deficient Chinese hamster ovary cells asdescribed previously (Cate, et al. Cell, 45: 685-698 (1986)).Alternatively, a mutated cDNA which produces a bioinactive form of humanMIS was also cloned into the CHO cells, resulting in the CHO-L9 cellline (Kurian et al, Clin. Cancer Res. 1(3):343-349 (1995). Both celllines were maintained in DMEM supplemented with glutamine, penicillin,streptomycin, methotrexate, and 5% MIS-free female fetal calf serum.

Subrenal Capsule Assay

Following the method of Bogden et al. Exp Cell Biol 47(4):281-93 (1979),as modified by Fingert, et al. Cancer Res., 47: 3824-3829 (1987), theIGROV-1 tumor cell line was tested for growth in vivo in a murinesubrenal capsule assay (Donahoe et al, 1984). Ten million cells werecentrifuged at 1500 rpm for 5 min to form a pellet. 300 micrograms offibrinogen (Sigma; 20 mg/ml, dissolved in phosphate-buffered saline, pH7.4) were added to the pellet, followed by 0.16 units of thrombin(Sigma; 20 unit/ml, dissolved in double-strength DMEM). This mixture wasincubated at 37° C. for 15 minutes. The cell clot thus formed was thencut into approximately 50 fragments (10 microliter volume, 200,000 cellseach) in preparation for implantation. Selected fragments were dissolvedwith trypsin and cells counted to confirm uniformity of cell number.

After inducing anesthesia using ketamine/xylazine (100/10 mg/kg BW,i.m.) a subcapsular space was developed in the left kidney with a19-gauge needle trocar and a cell clot measuring approximately 1×1ocular micrometer units at 7×magnification was introduced. The longestdiameter (L1) of the implant and the diameter perpendicular to thelongest diameter (W1) were measured with the ocular micrometer of adissecting microscope. The graft volume was estimated as L1×W1×W1. Theimplant was allowed to grow for 2-3 weeks at which time similarmeasurements were obtained at the same focal distance as the initialmeasurement to calculate the graft size ratio ([L2×W2×W2]/[L1×W1×W1]).Histology of the tumors was reviewed to verify that the implants wereviable and lacked both an inflammatory infiltrate and central necrosis.The growth of the tumor was measured at weekly intervals and the time ofthe experiment chosen so that 3 to 4×growth was achieved for thecontrol-treated tumors.

Preparation of the Polymer-cell Graft and its in vitro Production of MIS

Biodegradable polymer consisting of 1 millimeter thick sheets ofnonwoven fibers of polyglycolic acid (density 70 mg/cc, fiber diameter14 μm, and average pore size 250 μm) was obtained from Smith and Nephew(York County, UK). The sheets were sectioned into 0.5 cm² squares whichwere placed in a 12-well tissue culture plate (Costar, Cambridge,Mass.), sterilized with 95% ethanol, and washed with phosphate buffersaline (PBS). Sterile filtered IN sodium hydroxide was added to eachwell for 60 seconds to make the polymer hydrophilic and the polymer waswashed with distilled water and coated with collagen (Vitrogen 3 mg/mldiluted 100× in sterile PBS) added to the wells for one hour at roomtemperature. The treated polymer squares were further washed withsterile PBS. Transfected CHO cells were remove from culture flasks withtrypsin-EDTA (Gibco) and resuspended in DMEM supplemental with 10%female fetal calf serum. After counting the cells in a Coulter counter,1-2×10⁶ suspended cells were seeded with a micropipet onto each polymersquare and absorbed onto the interstices of the polymer matrix during asubsequent incubation of 1-2 hours at 37° C. in 5% CO₂ air to allowsufficient time for attachment, after which time fresh media was addedand the wells returned to the incubator for attachment, after which timefresh media was added and the wells returned to the incubator for threeto seven days. Growth media was changed every other day. Theconcentration of MIS in the media was measured serially using asensitive MIS ELISA (Hudson et al, 1990) and an MIS specific organculture assay (Donahoe 1977) was used to assess MIS bioactivity onselected samples.

Implantation of polymer-cell graft and the IGROV-1 tumor cell line intoSCID mice

On day 0 the cells are seeded onto the polymer. 3-7 days later, when themedia MIS levels reach 200 ng/ml, the polymer-cell graft is implantedinto the right ovarian pedicle of SCID mice. On day 10-14, the IGROV-1tumor in the form of a cell clot is implanted under the left renalcapsule of the mice. 2-3 weeks after implantation, the size of thetumors is measured. The left kidney and the right ovarian pedicle areremoved for immunohistochemical and/or histologic analysis. Serum iscollected throughout the protocol to determine MIS concentrations.

Three to seven days after seeding with CHO-B9 or CHO-L9 cells, whenserum MIS was above physiologic levels, a 5×5×1 mm polymer square wasimplanted into the right ovarian pedicle of SCID mice as described fortumor samples by Kristjansen, et al. Microvasc. Res., 389-402 (1994).After induction of anesthesia with ketamine/xylazine, a one centimeterhorizontal incision was made in the right flank. The ovarian pedicle wasidentified, delivered out of the wound, and the polymer-cell graft laidon the ovarian pedicle and sutured in place with 6-0 prolene. Six tothirteen days later, the levels of circulating MIS were determined. Whenthey approached supraphysiologic levels, the IGROV-1 tumor cell line wasprepared in a fibrin/thrombin cell clot and implanted under the leftrenal capsule as described above. Different sized polymers (0.125, 0.25,0.5, and 1.0 cm²) seeded with CHO-B9 cells were implanted into SCID miceand serum MIS levels measured to determine the optimal size of thepolymer implant. Two to three weeks after implantation of the IGROV-1cell clot, the left kidney was exposed and the dimensions of theimplanted tumor measured. The graft size ratio was calculated andcomparisons made between groups of animals receiving the B9, L9, orempty polymer. Also, at this time, the right ovarian pedicle (locationof polymer implantation) was removed and measured, and selected implantsexamined by immunohistochemistry or routine histology. The animalsimplanted with CHO-B9 seeded polymer served as the experimental groupand the animals implanted with CHO-L9 seeded or empty polymer served ascontrols.

Serum MIS levels and bioassay

MIS was measured at various time points after polymer implantation usinga human MIS-specific ELISA described previously (Hudson et al, 1990).MIS-containing serum was placed in the MIS organ culture assay (Donahoeet al, 1977) to correlate bioactivity of the MIS present in the sampleto the MIS levels as measured by ELISA.

Tissue Analysis

The tissue formed from the cell-polymer implant in the right ovarianpedicle and the kidneys with implanted IGOV-1 cell clot were fixed in 5%picric acid and 15% formalin in PBS. The tissue was then processed andcut into 8 micron sections prior to staining either for routinehistologic analysis or for immunohistochemistry (Gustafson et al,N.Eng.J.Med. 326(7):466-471(1992). Viability of the tumors was confirmedas they were evaluated for central necrosis or an inflammatoryinfiltrate. Selected ovarian pedicles were harvested earlier during thecourse of the experiment and examined histologically to determine therate of biodegradation of the polymer.

Statistics

Values for the tumor graft-size ratio are expressed as mean +/− standarderror (SE). An unpaired t-test performed by ‘STATVIEW’ and analysis ofvariance (AVOVA) performed by ‘EXCEL’ were used to determine the levelof statistical significance (p values).

Results

Production of MIS in vivo

The polymer-cell graft was incubated in vitro for three to seven days,at which time MIS levels of 100-400 ng/ml were measured in the media andthe graft was implanted into the ovarian pedicle of mice. Serum MIS wasmeasured by ELISA in the animals implanted with the different sizedpolymer squares (FIG. 2A). MIS levels in the animals implanted withpolymer squares measuring 1.0 (n=4) and 0.5 (n=4) cm² weresupraphysiologic at two weeks following implanation and exceeded 1microgram/ml 3 weeks after implantation. In the animals with smallersquares measuring 0.25 (n=4) and 0.125 (n=4) cm² the MIS levels weresupraphysiologic by three weeks following implantation. 0.5 cm2 wasselected as the optimal size of seeded graft for implantation. The CHOcell line would not grow in animals made immunosuppressed byinactivation of the RAG-2 gene, hence the SCID mouse where growth andMIS production were robust was selected.

Serum MIS was measured by ELISA at 7, 14, 21, and 28 daysafter-implanatation of a polymercell graft measuring 0.5 cm² (n=40). At14 days, the levels of MIS measured 100-500 ng/ml and at 28 days thelevels measured 7-10 micrograms/ml (FIG. 2B). When the polymer-cellimplant was removed, the serum levels were undetectable within 7 days(n=1) (FIG. 2C).

Sera from mice with high MIS levels were analyzed in the MIS bioassay todetermine the bioactivity of the MIS produced by the polymer-cellimplant. The samples produced complete regression of the Mullerian ductin the organ culture assay, indicating that the MIS in the serum ofanimals retained bioactivity. The MIS produced in vivo is bioactive.When placed in an organ culture assay, serum from the animals with aCHO-B9 polymer-cell graft implant causes complete regression of the ratMullerian duct, leaving only the Wolffian duct. The negative controlculture shows both Mullerian and Wolffian ducts.

After two weeks in vivo, the polymer-cell graft formed a firm, livingmass of tissue throughout the polymer fibers, which began resorbing.After 4 weeks in vivo, the biodegradable polymer could no longer bedetected and the polymer-cell graft grew into a rounded, well-containedmass with an approximate diameter of 1.0 cm. There was no evidence ofspread of CHO-B9 cells beyond the mass formed in the ovarian pedicleduring the duration of the experiment. The mass consisted histologicallyof epithelial cells with evidence of ingrowth of blood vessels from theovarian pedicle. Immunohistochemical analysis confirmed the cellsgrowing on the polymer continued to synthesize MIS. Immunohistochemistryof the polymer-cell graft after 4 weeks in vivo indicates ongoingproduction of MIS by the implanted cells. The polymer-cell graft stainedwith an antibody to human MIS is in marked contrast to the stainingpattern seen using a control antibody.

Inhibition of tumor growth by MIS produced by polymer-cell implant

The IGROV-1 tumor cell line, when implanted into the subrenal capsule ofthe SCID mice, formed measurable tumors growing to reach a volumegraft-size ratio of 3-4 three weeks after implantation. Histologicanalysis of these tumors demonstrated well-formed growths withneovascularization from the underlying kidney parenchyma. There wasminimal necrosis and inflammatory infiltrate. After 3 weeks of exposureto MIS, the tumor is approximately one third the size of the control andalso has minimal necrosis or inflammation. When the IGROV-1 tumor cellline was implanted under the renal capsule of nude athymic mice, thetumors failed to achieve 3-4 fold growth three weeks after implantation.Hence the SCID mice were selected for use in subsequent experiments.

Animals implanted with the bioactive MIS-producing CHO-B9 polymer (n=8)showed very little net growth of the IGROV-1 implant, achieving a meangraft-size ratio +/− the standard error of the mean of 1.62+/−0.218(FIG. 3A). The tumors implanted into the bioinactive MIS-producingCHO-L9 polymer (n=10) achieved a mean gift-size ratio of 3.25+/−0.506.This difference was statistically significant with a p-value of 0.016(FIG. 3A).

Three experiments were performed in which animals were implanted withthe bioactive MIS-producing CHO-B9 polymer or polymer without cells. Inthe first experiment the graft-size-ratios of IGROV-1 tumors were1.323+/−0.168 (n=10) for the animals with CHO-B9 polymer and3.427+/=0.682 (n=10) for the animals with polymer alone. In the secondexperiment the graft-size-ratio of the tumors in animals implanted withCHO-B9 polymer and polymer alone were 2.025+/−0.198 (n=10) and3.024+/−0.454 (n=10). In the third experiment the graft-size-ratio ofthe tumors in animals implanted with CHO-B9 polymer and polymer alonewere 1.427+/−0.147 (n=10) and 2.447+/−0.375 (n=10). The averagegraft-size ratios for all three experiments combined were 1.592+/−0.112in the CHO-B9 polymer animals and 2.966+/−0.299 in the animals withempty polymer (FIG. 3B). The difference in graft-size-ratio between thetwo groups was statistically significant with a p-value <0.001.

In summary, cells in this study were transfected with the gene encodinighuman MIS, seeded onto biodegradable polyglycolic acid fibers, and shownto produce biologically active MIS in vitro. When the polymer-cellgrafts were implanted into animals, the continually produced MIS couldbe detected in the serum of the animals within one week of implantation(FIGS. 2A, B). Over time, secreted MIS was detected by ELISA inincreasing concentrations, and the serum, when tested in the standardMIS in vitro bioassay, was biologically active (FIGS. 3A,B). There wereno adverse effects in the animals as a result of the either the presenceof the polymer, the growing cells, or the high levels on MIS. Removal ofthe polymer-cell graft resulted in declining and, after one week,undetectable levels of MIS (FIG. 2C) suggesting no spread of cells fromthe ovarian pedicle. When tumor cell line responsive to MIS in vitro wasimplanted in the subrenal capsule of the mice containing the MISsecreting graft, the growth of the target tumor was slowed considerably.The graft-size ratio of the measured tumors was significantly smallercompared to the growth of tumors implanted in animals with a polymersecreting biologically inactive MIS or a polymer secreting no MIS (FIGS.3A, 3B). Thus only bioactive MIS and not a CHO cell product orbiopolymer component was responsible for the growth inhibition.Histologic analysis confirmed the three-dimensional growth of the tumorsand demonstrated lack of necrosis or excess inflammation that couldalter the size of the controls.

Example 3 rhMIS Production by Genetically Engineered, AutologousFibroblasts

To be used clinically, MIS must be administered to patients in a safeand cost effective manner in sufficient quantities to achieve tumorinhibition. MIS is a molecule that is produced by the Sertoli cells ofthe testis in the male and the granulosa cells of the ovary in thefemale. It serves the known function of Mullerian duct regression in thedeveloping male fetus and likely serves as a modifier of cell growth anddifferentiation in the male and female throughout life. MIS is a complexmolecule consisting of 70 kDa homodimer subunits that requires proteasemediated cleavage for biological activity; therefore, production anddelivery of purified MIS is predicted to be a complicated and costlyendeavor. As an alternative to production of the purified protein, theuse of biodegradable polymer seeded with MIS-producing transfected cellscan deliver biologically active MIS in quantities sufficient to achieveserum levels above the highest measured in newborn males, while avoidingcomplicated purification protocols.

The studies described herein demonstrate proof of principle for thedevice using a partially transformed Chinese hamster ovary epithelialcell line that can be tumorigenic in immunosuppressed mice. Normal humanfibroblast cell lines and mouse fibroblasts harvested from theperitoneum of animals with rhMIS constructs have now been transfected.Wild type and more easily cleavable MIS (S428R, Kurian et al., 1995)constructs will be transfected into fibroblasts using state of the artstable transfection techniques optimized for transfection efficiency andMIS production followed by clonal selection of the cells that producethe greatest concentration of MIS. These will be seeded onto thebiodegradable mesh and implanted into mice and experiments will berepeated as with the CHO B9 cells. These experiments will be used toestablish the optimal geometry of the mesh that will produce the highestconcentration of MIS.

Human lung fibroblast cell lines IMR-90 will be permanently transfectedwith two monocistronic constructs encoding hygromycin resistance and oneof either pCDNA-vector or pCDNA-K2, a CMV-driven MIS ligand expressionconstruct. Transfection will be performed using either Fugene 6transfection reagent (Boehringer Mannheim) or using the standard calciumphosphate DNA precipitation technique. Cells will be plated in 100 cm²well plates. When they reach 60 to 80% of confluence, Fugene 6 at 2μg/ml will be added for 48 hours and washed. IMR-90 human fibroblastcells have been transfected using the Fugene system with 0.5 μghygromycin and 5 μg of the K2 constructs, as well as vector alone, andthese are now being selected in high concentration hygromycin media (750μg/ml of hygromycin, Boehringer Mannheim). After two weeks the mediawill be tested for MIS production by ELISA. Clones will be replated at10 cells/well in 24 well plates and expanded in media containing 100μg/ml of hygromycin. Clones will be selected for MIS production by ELISAof overlying media; high producers will be grown on biodegradablematrices and implanted in SCID mice harboring ovarian tumors. Primaryhuman fibroblasts originally taken from patients to study expression ofandrogen receptor will be similarly tested for transfection efficiencyusing retroviral transfection and then clonally selected for maximumproduction of MIS.

Since primary fibroblasts are difficult to transfect, an adenovirustransfection system adapted from one that used to transfect primarySertoli cells will be used. Briefly, 32.5 μl of adenovirus, diluted inPBS with 10% glycerol and 0.2% BSA, is added to the fresh medium andincubated for 1 hour at 37 C. After 1 hour of incubation, cells will bewashed with HBSS and added in 500 μl of fresh medium. 48 hours latercells the media will be collected and assayed for MIS using the MISELISA and the organ culture assay for regression of the Mullerian Duct.The IMR-90 cells in 24 well plates have been transfected with a CMV-GFPvirus and detected near 100% infection by fluorescence. Three otherhuman fibroblast lines followed by primary fibroblasts will betransfected, then virus to express the MIS construct generated.

Since growth after transfection and cloning may be inefficient infibroblast cell lines and primary fibroblasts, clones of cells may haveto be screened before choosing the best MIS producers to seed the mesh.An alternative approach is to grow pools of cells ex vivo on individualbiodegradable meshes and then to test and select each mesh for maximalproduction of MIS prior to implantation in vivo for serial measurementsof MIS in each animal's serum. Once the method of “cloning” in the meshor in monolayer culture is established, and the highest ex vivoproducers are selected, the loaded meshes will be implanted in anovarian fat pad and levels of MIS production measured and compared tothe levels produced by CHOB9 cell impregnated implants or by MIScontaining Alzet pumps.

The next step is to implant tumors in the subrenal capsule of 20 SCIDmice for as many days as it takes for the tumor to reach 4 times theoriginal implant volume measured at length×width×width at the time ofimplantation (Parry et al., 1992). The time to reach 4× for each cellline and the time to reach maximum production per unit number of cellsand unit size of mesh will be determined, then after the mesh isproducing high levels of MIS the tumors will be implanted. Growthbetween mesh containing MIS and non MIS producing fibroblasts (n=20) andAlzet delivered MIS positive controls (n=20) will then be compared.Subsequently tumor growth will be allowed to reach 3-4× in size. ThenMIS producing versus non-producing fibroblast-containing mesh will beimplanted intraperitoneally and growth of tumors compared between groupsto see if MIS prevents growth and/or reverses growth of the tumorimplants.

The biodegradable mesh impregnated with autologous fibroblasts will thenbe implanted into patients. It is important to note that either dermalfibroblasts, peripheral or marrow stem cells, or peritoneal mesotheliumwill be requested from patients. The autologous cells will then betransfected and cloned and the optimal MIS producing fibroblasts will beimpregnated in the biodegradable matrix plugs. The matrices will then beimplanted into the ovarian pedical in the peritoneal cavity of theovarian cancer patient from whom the fibroblasts were taken. This mayentail implantation in or near the tumors intraperitoneally for theovarian cancer model or in the liver, brain, heart, blood vessels,joints, or other organs, as the protein of interest or therapeuticindication dictates.

This ongoing work uses transfected fibroblasts which grow robustly onthe polymer. To avoid immunosuppression in patients, a sample of thepatient's own cells will be transfected with the human MIS genesequence. The cells could be fibroblasts or myofibroblasts obtained froma small skin or muscle biopsy or stem cells from peripheral blood orbone marrow. The cells would be grown on biodegradable polymer in vitroand implanted in the patient, providing continual production of MIS toserve as an inhibitor of tumor growth.

Modifications and variations of the present invention are intended tocome within the scope of the following claims.

We claim:
 1. A method for treating abnormal cell or tissue growth thatis treatable with MIS, in a subject in need thereof, comprisingimplanting in the subject a cell-matrix structure, wherein thecell-matrix structure comprises a matrix having attached theretoallogeneic or autologous cells that have been genetically engineered tostably express MIS in an amount sufficient to treat the abnormal cell ortissue growth, and wherein the abnormal cell or tissue growth is vulvarepidermoid carcinoma, cervical carcinoma, endometrial adenocarcinoma,ovarian adenocarcinoma, or a breast tumor.
 2. The method of claim 1wherein the abnormal cell or tissue growth is.
 3. The method of claim 1wherein the matrix is selected from the group consisting of fibrousscaffolds, polymeric hydrogels, and micromachine or micromoldedsubstrates.
 4. The method of claim 1 wherein the cells that have beengenetically engineered are of a different cell type than the abnormalcell or tissue.
 5. The method of claim 1 wherein the abnonnal cell ortissue growth is ovarian adenocarcinoma.
 6. The method of claim 1wherein the abnormal cell or tissue growth is malignant.
 7. The methodof claim 1 wherein the tissue is ovarian tissue.
 8. The method of claim1 wherein the matrix comprises a biodegradable polymer of polyglycolicacid.